PN-2 is believed to be derived from .beta.-amyloid precursor protein (hereinafter ".beta.APP"). As used herein, ".beta.APP" shall be used to designate the unprocessed, non-secreted forms of this amyloid precursor protein. Alternatively spliced forms of the two major mRNA transcripts for .beta.APP produce an unprocessed, non-secreted, membrane bound or possibly intracellular, full length protein of either 695 or 751 amino acids. Thus ".beta.APP.sub.695 " and ".beta.APP.sub.751 " are used herein to designate the 695 and 751 amino acid forms of the protein, respectively. .beta.APP.sub.751 includes a 56 to 57 amino acid residue insert which is homologous to that of a "Kunitz-type" protease inhibitor which inhibits trypsin.
Each of the two major forms of .beta.APP is processed to form the two major secreted forms. It is the major secreted form of the 751 amino acid translation product which is referred to herein as "PN-2". The secreted form of the 695 amino acid protein is referred to herein as "APP.sub.695sec ".
For reasons which are discussed hereinbelow, the term "PN-2/.beta.APP" is used herein generically to refer to all proteins having sequence homology with the amyloid precursor protein.
Alzheimer's Disease (AD) produces a debilitating dementia for which no treatment is known, with no known reliable methods of diagnosis prior to autopsy. .beta.APP and its various processed forms are known to be associated with neural lesions of AD, as well as with Down's syndrome. AD is characterized by the accumulation of amyloid protein both intracellularly and extracellularly in the tissues of the brain, notably in neuritic plaques. The major protein subunit of these amyloid plaques has been identified as a polypeptide of about 4.5 kD having the ability to aggregate. This protein subunit is variously referred to as the amyloid .beta.-protein or as amyloid A4, and is herein referred to as "A4".
The cDNA corresponding to the mRNA for the 695 amino acid .beta.APP lacking the Kunitz domain has been cloned and the nucleotide sequence determined. From the nucleotide sequence, the amino acid sequence has been predicted. The A4 peptide lies at residues 597 to 648 of the deduced amino acid sequence of .beta.APP.sub.695.
A4 is thought to have its origin, through proteolytic cleavage, in .beta.APP. Release of the A4 unit is thought to occur by proteolysis of the precursor which may result from membrane damage. Because A4 is believed to be critical to the formation of amyloid plaques, there is a need for methods of preventing the release of A4.
Down's syndrome is a genetic disease which usually causes mental retardation and other symptoms. An unusually high number of people with Down's also develop Alzheimer's after the age of 40. Thus, there is a need for a treatment for Down's.
As discussed above, definitive diagnosis of Alzheimer's disease is only available at autopsy. Such diagnosis involves examination of brain tissue for extracellular neuritic plaques and intracellular tangles of microtubule-associated proteins and other cytoskeletal elements. The plaques are believed to start to form years before any clinical sign of Alzheimer's appears.
Many researchers believe that there is a correlation between the density of neuritic plaques and the severity of dementia. Thus, there is a need for preventing further development of neuritic plaques. If applied prior to the development of Alzheimer's symptoms, such a treatment, may thereby prevent onset of the disease.
Even without a treatment for Alzheimer's, an early diagnosis of the disease would allow physicians to rule out other causes of dementia. Moreover, in order to study the genetics of Alzheimer's it would be very useful to obtain a diagnosis of the disease prior to autopsy. More importantly, once treatments are developed, an early diagnosis may prove critical in the treatment's ability to improve mental functioning. Thus, there is a need for a method for the early diagnosis of Alzheimer's.
Currently, there is no reliable biochemical test available to assist in the diagnosis of Alzheimer's disease in live patients. Previous studies have produced conflicting data regarding the usefulness of measuring PN-2/.beta.APP in cerebrospinal fluid ("CSF") to assist in diagnosis of Alzheimer's disease. Three such studies (Kitaguchi et al., Biochem. Biophys. Res. Comm. 166:1453-1459 (1990), Weidemann et al., Cell 57:115-126 (1989) and Ghiso et al., Biochem. Biophys. Res. Comm. 163:430-437 (1989)) suggested that increased levels of PN-2/.beta.APP immunoreactivity can be found in the CSF of AD patients compared with controls. Another study (Palmert et al., Neurology 40:1028-1034 (1990)) found that the absolute and relative levels of PN-2/.beta.APP in cerebrospinal fluid overlapped between Alzheimer's disease patients and a control group. Yet another study (Henriksson et al., J. Neurochem 56:1037-1042 (1991)) indicated that while PN-2/.beta.APP levels were slightly reduced in AD relative to control, that measurement of PN-2/.beta.APP in CSF did not appear to be useful in diagnosing Alzheimer's disease.
Still another study (Prior et al., Neuroscience Letters 124:69-73 (1991) indicated that the average amount of .beta.APP immunoreactivity in CSF was lower in a group of AD patients relative to a group of control subjects and was only slightly lower than a group of multiple infarct dementia patients. However, in this study, the range within each of these groups was very broad and overlapping between groups. Thus, this study failed to provide an indication of the presence of AD for any particular individual.
All of the foregoing studies of PN-2/.beta.APP immunoreactivity in CSF used polyclonal or monoclonal antibodies raised against synthetic proteins. These monoclonal antibodies lacked sufficient sensitivity to reliably determine differences in immunoreactivity among samples containing high levels of protein, such as CSF samples. The use of polyclonal antibodies results in cross-reactivity with other proteins. Thus, quantitation can only be achieved with these antibodies by performing immunoblots and excising major bands. The cross-reactivity of these antibodies with proteins other than PN-2/.beta.APP prevents them from being effectively used in enzyme-linked immunosorbent ("ELISA") assays. In light of the inability of previous studies to reliably detect Alzheimer's Disease, there remains a need for a reliable test to assist in the diagnosis of Alzheimer's Disease in live patients.